Many proteins, lipids and others exist in living bodies. Although they may exist by themselves, previous studies have revealed that in many cases they are somewhat modified to function properly in the living body. For example, phospholipids are kinds of lipids each of which has a phosphoric ester as a partial structure, and mainly exist as a major component of cell membranes in the living body. Some phospholipids are known to have bioactivities such as the involvement in signal transduction in the living body. Further, the alterations of the phospholipids in the blood or a body tissue has been found to be associated with various diseases.
As shown in FIG. 3A and FIG. 3B, phospholipids are classified roughly into two kinds: glycerophospholipid having glycerin as a central skeleton and sphingophospholipid having sphingosine as a central skeleton. As shown in FIG. 3A, glycerophospholipid has a structure in which a fatty acid and a phosphate group are bonded to glycerin as the central skeleton and further a base such as choline, ethanolamine, inositol and serine is bonded through the phosphate group. On the other hand, sphingophospholipid has a structure in which a fatty acid, a phosphate group and the like are bonded to sphingosine as the central skeleton. Glycerin, sphingosine and the fatty acid are hydrophobic portions, the phosphate group and the base are hydrophilic portions, and phospholipids are amphiphilic substances each of which has the hydrophobic portions and the hydrophilic portions in a single molecule.
Theoretically, a wide variety of phospholipids can exist depending on the difference in the hydrophilic polar group portion that is a characteristic partial structure for discriminating the kind of the phospholipid (for example, phosphatidylcholine, sphingomyelin and the like described in FIG. 3A and FIG. 3B), the difference in the carbon number, double bond number and bonding site of the bonded fatty acid, and various combinations of bonding types (ester type, ether type and alkyl type) between the fatty acid and the central skeleton, and the like. In the simultaneous analysis of the phospholipids using a mass spectrometer, the related art employs a technique of focusing on the difference in the characteristic partial structure for discriminating the kind of the phospholipid and detecting a molecular ion associated with the desorption of a site corresponding to the characteristic structure, that is, the desorbed site itself, or a technique of detecting, to the contrary, a molecular ion corresponding to the remaining structure after the desorption.
For example, Patent Literature 1 describes that selection is made based on the polar group portion (hydrophilic portion) of the lipid at an MS2 stage by a neutral-loss scan measurement with the collision-induced dissociation (CID) under a collision energy specific to the lipid species of an analysis object, and further an exhaustive analysis of the lipid is performed at an MS3 stage by the analysis of the aliphatic hydrocarbon chain that is the hydrophobic portion of the lipid.
As an exhaustive analysis using a multiple reaction monitoring (MRM) measurement in a tandem quadrupole mass spectrometer, it has been reported that the kind of a disease-specific phospholipid in a biological sample has been specified by setting, as an analysis condition, an MRM transition (a combination of the m/z values of a precursor ion and a product ion) corresponding to each phospholipid class (molecular species), using the structural characteristic of each phospholipid class (see Non Patent Literature 1). However, in such an MRM measurement using the characteristic partial structure of the phospholipid class, the structural information of the fatty acid constituting the phospholipid cannot be obtained. Therefore, by concurrently performing an MRM measurement for fragment ions of the fatty acid based on the desorption, the structural information of the fatty acid of a particular phospholipid is acquired together, and the identification of the phospholipid is performed. In this case, since the class of the phospholipid as the analysis object is originally limited, the total number of combinations between MRM transitions corresponding to the phospholipid classes and MRM transitions corresponding to fatty acid structures is not very large.
Generally, in a liquid chromatograph (LC) or gas chromatograph (GC) in which the tandem quadrupole mass spectrometer is adopted as a detector, a preset measurement sequence in which structure-specific MRM transitions are variously combined corresponding to the retention tune is used to perform the simultaneous multicompound analysis by a single LC/MS analysis or GC/MS analysis for a target sample. However, in the case of the phospholipid, the number of MRM transitions for determining the phospholipid class is 400 or more, and the number of MRM transitions for determining the fatty acid composition is 800 or more. Therefore, when the simultaneous multicompound analysis is performed while the class of the phospholipid as the analysis object is not previously limited, it is substantially impossible to arrange the measurement sequence such that all phospholipids can be identified by a single LC/MS analysis, even when MRM transition combinations different for each retention time are assigned after the phospholipids are separated by the LC. Further, in the case of the phospholipid, there is a circumstance in which the switching between a positive-ionization mode and a negative-ionization mode needs to be performed depending on the target substance (partial structure) in the MRM measurement and it is difficult to execute them in an identical measurement event (a determined measurement time range for repeatedly executing measurements different in the measurement condition including the MRM transition, by time division).
Hence, the simultaneous multicompound analysis of phospholipids needs to be executed in the following procedure. Specifically, the LC/MS analysis is first performed for the target sample while MRM transitions for determining the phospholipid class are adopted as measurement conditions, whereby one or more classes of the phospholipids contained in the sample are specified. Thereafter, only for the previously specified phospholipid classes (that is, the classes of the phospholipids confirmed to be contained in the target sample), the LC/MS analysis is performed for the same target sample while MRM transitions for determining the fatty acid composition are adopted as measurement conditions. Then, the phospholipids in the target sample are identified based on the results of the two LC/MS analyses.
In the case of such a two-stage LC/MS analysis, if the MRM transitions in the first-time LC/MS analysis and the MRM transitions in the second-time LC/MS analysis have a one-to-one correspondence relation, the creation of the measurement sequence in the second-time LC/MS analysis is relatively easy. However, the correspondence relation between the MRM transitions for determining the phospholipid class and the MRM transitions for determining the fatty acid composition is complex. To create the measurement sequence in the second LC/MS analysis, it is necessary to select MRM transitions for confirming the fatty acid composition that can correspond to the specified phospholipid classes from an enormous number of combinations of phospholipids, which is very cumbersome and troublesome. Further, the number of the MRM transitions for determining the fatty acid composition that corresponds to the MRM transitions for determining the phospholipid class varies, causing a problem in that an error is prone to occur in the creation of the measurement sequence and such an error may be left unnoticed.
Such a problem is not limited to phospholipids, and a similar problem can occur, for example, in peptides after posttranslational modification in which posttranslational modifying substances such as carbohydrate chains are bonded to peptides having various lengths (amino-acid sequence lengths), and compounds in which partial structures bonded to the basic skeleton are variously changed by metabolism or intentional modifications.